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Lecture 2
• Overview of preimplantation development
• Specification of the trophectoderm
• Specification of primitive endoderm
• Stem cell lines from early mouse embryos
You should understand
• Key transcription factors and signalling pathways in preimplantation embryos
• Mechanisms governing specification of the trophectoderm lineage
• Mechanisms governing specification of the primitive endoderm lineage
• Stem cell lines from early mouse embryos and their relationship to early
lineages.
Mosaic and Regulated development
•
Roux (1888) shows ‘mosaic development’ of frog embryo following ablation of one cell in
two-cell embryo – formation of ‘half’ embryo.
•
Driesch (1895) finds opposite is true for sea urchin, normal albeit smaller embryo develops
from one of two cells – ‘regulated development’.
Regulated development in mouse embryos
Donor
Recipient
2-cell
embryo
Tarkowski, (1959)
Nature 184, p1286-7
Chimeras from aggregaton of 8-cell stage embryos
8-cell embryos
Remove zona pellucida
Aggregate in dish
Culture in vitro
Transfer to foster mother
Chimeric blastocyst
Chimeric progeny
Tarkowski (1961) Nature 190, 857-860
Chimeras from transfer of ICM cells
Gardner (1968), Nature 220, p596-7
• Gardner later showed fate of TE and PE is determined by blastocyst stage
Preimplantation Development
Cleavage
Morula
Blastocyst
Day 3.0
Day 3.5
Inner cell mass (ICM)
Zona pelucida
Day 4.0
Primitive ectoderm (PrEct)
Blastomere
Blastocoel cavity
Trophectoderm (TE)
Primitive endoderm (PE)
Four master transcription factors for early lineage determination
in preimplantation development
1. Oct4/Pou5f1; uniformly expressed in cleavage stages. Switched off in trophectoderm of blastocyst.
Knockout fails to develop ICM.
2. Cdx2; stochastically expressed from 8-cell stage. Progressively restricted to outer TE cells of
blastocyst. Knockout fails to develop trophectoderm.
3. Nanog; stochastically expressed from 8-cell stage. Switched off in TE. Expressed in salt and pepper
pattern in ICM eventually restricted to primitive ectoderm at d4. Knockout fails to develop ICM.
4. Gata6 (+Gata4); stochastically expressed from 8-cell stage. Switched off in TE. Expressed in salt
and pepper pattern in ICM eventually restricted to primitive endoderm at d4.
Double knockout fails to develop PE.
Cleavage
Morula
Blastocyst
Day 3.0
Day 3.5
Inner cell mass (ICM)
Zona pelucida
Day 4.0
Primitive ectoderm (PrEct)
Blastomere
Blastocoel cavity
Trophectoderm (TE)
Primitive endoderm (PE)
Inside-Outside Hypothesis
8-cell embryo
16-cell compacted morula
Outside cell
Inside cell
Tarkowski and Wroblewska, (1967) J Embryol Exp Morphol. 18, p155-80
Testing the inside outside hypothesis
4-cell
embryo
Hillman, Sherman, Graham (1972) J. Embryol. Exp. Morphol. 28, 263-278
The role of compaction and the cell polarity model
• Compaction; at 8-cell stage cells flatten along basolateral surfaces (those with cell-cell contacts).
Apical (outside facing) surfaces develop distinct features, eg microvilli.
• Cell polarity model posits that divisions at 8-cell stage produce 2 polar or 1 polar and one apolar
cell, depending on the plane of division (stochastic).
Johnson and Ziomek (1981), Cell 21, p935-942
Cell polarity at compaction discriminates outer
and inner cells of the morula
8-cell
compaction
Basolateral
determinants
16-cell morula
Apical determinants
Non-polar
Inside cell
Polar outside cell
• Only outside cells express apical determinants – provides potential mechanism for
the differentiated fate decision.
Molecular mechanism linking polarity to TE specification?
•
Proteins of the apical-basal polarity pathway localise assymetrically in the morula
Inhibition of Hippo signalling in polarised cells induces Cdx2
•
Tead4, the downstream effector of Hippo pathway is required for Cdx2 expression in outer cells.
•
Tead4 co-activator, dephosphorylated YAP is present in the nucleus only in inner cells of 32 cell morula.
Nishioka et al (2009) Dev Cell 16, p398-410
Maintenance of TE/ICM specification
•
Double negative feedback loop with Oct4/Nanog confines Cdx2 expression to TE cells.
Specification of primitive endoderm lineage
Day 3.0
Day 3.5
Inner cell mass (ICM)
Blastocoel cavity
Trophectoderm (TE)
Day 4.0
Primitive ectoderm (PrEct)
Primitive endoderm (PE)
High Nanog
Low GATA6
Low Nanog
High GATA6
•
Reciprocal salt and pepper pattern of Nanog and GATA6 in ICM cells of mid-stage blastocysts
Chazaud et al (2006) Dev Cell 10 p615-24.
Fibroblast growth factor (FGF) signalling transduced
by MAPK
• Grb2 mutant embryos fail to specify primitive endoderm
Chazaud et al (2006) Dev Cell 10 p615-24.
Fibroblast growth factor (FGF) signalling regulates
primitive endoderm to primitive ectoderm switching
Fgf4
Nanog
Grb2
Fgf2r
Fgf4
Mapk
Gata6
Nanog
Gata6
Fgf4 high
Fgfr2 high
Primitive
ectoderm
(PrEct) cell
Primitive
endoderm
(PE) cell
Cell sorting
•
FGF4 gene is activated by Oct4
•
Only Nanog expressing ICM cells seen in Grb2 knockout or with disruption of FGF signalling
•
Negative feedback by Gata6 on Nanog and vice versa?
•
Cell sorting mechanism?
Chazaud et al (2006) Dev Cell 10 p615-24.
Embryonic Stem (ES) Cells
Stem cells and progenitors;
Stem cell; unlimited capacity to self-renew
and produce differentiated derivatives
Progenitor cell; limited capacity to self-renew
and produce differentiated derivatives
Terminally differentiated cell
Terminology for differentiative capacity of stem cells/progenitors;
• Totipotent; capable of giving rise to all differentiated cell types of the organism,
including extraembryonic lineages e.g. morula cells
• Pluripotent; capable of giving rise to cell types of the three germ layers, ectoderm, mesoderm
and endoderm eg primitive ectoderm cells of the blastocyst.
• Multipotent – capable of giving rise to a limited number of differentiated cell types, e.g.adult
stem cells and progenitors
ES cells
• Derived from blastocyst stage embryos
• Grow as ‘clumps’ or ‘colonies’ by culturing with fetal calf-serum (FCS) on layer of inactivated
primary embryonic fibroblast cells (PEFs).
Alkaline phosphatase positive
Core transcription factors Oct4, Nanog and Sox2.
• Have stable karyotype and contribute to all three germ layers (but not trophectoderm)
when transferred to recipient blastocyst – pluripotent.
• Contribute to the germ-line of chimeric animals (blastocyst injection) and can therefore be
transmitted to subsequent generations.
• Can be differentiated into embryoid bodies or defined lineages in vitro
Evans and Kaufman (1984) Nature 292, p154-6
What is an ES cell?
•
Single cell transcriptomics suggest closest to primitive ectoderm cells of the blastocyst.
•
No self-renewing pool of embryonic precursors in ICM or epiblast – ES cells are ‘synthetic’.
Signalling pathways regulating self-renewal
and differentiation of mouse ES cells
LIF/STAT3 (JAK/STAT)
and BMP/Smad/Id
GSK inhibition
(wnt?)
FGFs
Via ERK1/2 pathway
LIF/STAT3 and
BMP/Smad/Id
•
2i - Small molecule
inhibitors of ERK
GSK inhibition
(wnt?)
Recent evidence suggests LIF +BMP blocks autostimulation of differentiation by FGF4
Ying et al (2008) Nature 453, p:519-23
Stem cell types isolated from early mouse embryos
Day 4.0
Day 3.5
Polar Trophectoderm
ICM
Mural Trophectoderm
+FGF4
-LIF
+ feeders
+LIF
+BMP
ES cell
Day 5.5
Extraembryonic
ectoderm
Visceral
endoderm
Polar Trophectoderm
Primitive ectoderm
Primitive endoderm
Mural Trophectoderm
Parietal
endoderm
Epiblast
+FGF4
+LIF
+ feeders
+FGF
+Activin
TS cell
XEN cell
EpiSC
(Trophoblast
stem cell)
(Extraembryonic
endoderm cell)
(Epiblast stem cell)
Germ layers
Germ line
Trophectoderm
Primitive endoderm
Germ layers
Germ line
Trophectoderm
Primitive endoderm
Chimera
Contribution
Germ layers
Germ line
Trophectoderm
Primitive endoderm
Germ layers
Germ line
Trophectoderm
Primitive endoderm
In vitro
differentiation
(-LIF/-BMP)
Germ layers
Germ cells
Primitive endoderm
(-FGF)
Trophoblast giant
cells)
(-FGF)
Parietal endoderm like
(-FGF/Activin)
Germ layers
Tanaka et al (1998) Science 282, p2072-5; Brons et al (2007) Nature 448, p191-5;
Kunath et al (2005), Development, 132, p1649-61
Interconversion of embryo stem cell types
XEN
+GATA6
and/or
+OCT4
+FGF4
+LIF
ES
+CDX2
and/or
-OCT4
TS
+FGF4
- LIF
+FGF2
+Activin
+serum free
medium
+LIF
+2i
Or
+KLF4
EpiSC
Niwa (2007) Development 134, p635-46
End lecture 2